GB2051081A

GB2051081A - Antigen for early pregnancy test and contraceptive vaccine

Info

Publication number: GB2051081A
Application number: GB8022626A
Authority: GB
Original assignee: Research Corp
Current assignee: Research Corp
Priority date: 1978-02-06
Filing date: 1979-02-02
Publication date: 1981-01-14
Also published as: GB2013690A; AU3987978A; SE8404250L; FR2416014A1; NL7809347A; GB2013690B; US4234561A; SE8404250D0; FR2416014B1; IT1101251B; BE871107A; GB2051080B; CA1113389A; JPS54105224A; DE2842612A1; GB2051081B; CH650593A5; JPS6340770B2; GB2051080A; SE7809249L

Description

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GB 2 051 081 A 1

SPECIFICATION

Antigen for early pregnancy test and contraceptive vaccine

This invention relates to antisera produced by novel antigens, which are useful in testing for pregnancy in humans and more particularly to antisera having an immunological reaction with human 5 chorionic gonadotropin, wherein the immunological cross-reactivity with human luteinizing hormone is1 5 reduced or eliminated. This invention also relates to the production of antigens useful for the preparation of the said antisera. The antigens may also be used for contraceptive purposes to terminate human pregnancy.

Human chorionic gonadotropin is a hormone produced by the placenta during pregnancy. 10 Presence of the hormone in the serum and urine therefore serves as an indication of pregnancy. The 10 presence of this hormone has been detected by its effect on the ovaries of animals and, more recently, by immunoassay.

However, because the tests used hitherto do not sufficiently distinguish between hCG and other hormones which are present, such as luteinizing hormone, the presence of hCG cannot be 15 unambiguously detected until several weeks after conception, by which time the levels of hCG are high 15 enough so that it can be detected even in the presence of interfering substances.

The use of sensitive procedures such as radio-immunoassay has not been practical because the antisera to hCG hitherto available have been reactive not only with hCG, but also with other hormones which are present, such as human luteinizing hormone (hLH), follicle stimulating hormone, and thyroid 20 stimulating hormone. This occurs because isoimmunization with hCG results in the production of 20

antibodies which cross-react with hLH, follicle stimulating hormone, and thyroid stimulating hormone.

These hormones resemble one another in consisting of two non-covalently bonded subunits, a and /}. Whereas the a-subunits are almost identical in all of them, the /5-subunits are hormone specific and are structurally dissimilar. Use of the /J-subunit of hCG (hCG-/5) as an antigen produces an antiserum having 25 improved specificity, but which still shows appreciable cross-reactivity with hLH. The molecular basis of 25 this cross-reactivity is the presence of considerable homology in the amino acid sequences of hCG-/5 and hLH-/3, especially in the amino terminal 75% of the molecule. The carboxy-terminal 32-residue peptide is unique to hCG-/3. The amino terminal 75% of the molecule contains 6 intrachain disulfide linkages which maintain the conformation of the molecule. This conformation is believed to be 30 responsible for much of the antigenic activity of hCG as well as its immunological cross-reactivity with 30 hLH.

In addition to its use as an indication of pregnancy, hCG is necessary for the maintenance of pregnancy. Hence, if the hormone can be neutralized, the pregnancy can be terminated. The neutralization of hCG can be the basis of a contraceptive vaccine. However, raising antibody to hCG in 35 humans presents two major problems. hCG is a human hormone, and humans normally will not produce 35 antibody to a human hormone. This limitation can be overcome by modifying hCG in such a way that it is not recognized by the human system as its own hormone and behaves as a foreign material. There is still another problem. The antibody to hCG would also neutralize another hormone, namely luteinizing hormone (LH) from the anterior pituitary gland, which is necessary for the normal human reproductive 40 cycle. This problem can be partially overcome by using one of the two components of hCG molecule, 40 designated as the /5-subunit, the antibody to which would predominantly neutralize hCG. However, it still lacks the desired specificity.

Pappenhagen et al, U.S. Patent 3,903,262, disclose modifying serum globulins by reducing and cleaving disulfide bonds and subsequently alkylating the cleaved disulphide bonds for the purpose of 45 reducing the anticomplement activity of such globulins. However, Pappenhagen does not disclose 45

modifying serum globulins for the purpose of obtaining antibody selectivity for one hormone over another, nor does he disclose conjugation of globulins with other proteins.

The procedure of conjugating the /5-subunit of human chorionic gonadotropin with tetanus toxoid to reduce cross-reactivity with other hormones, including follicle-stimulating hormone, thyroid-50 stimulating hormone, and luteinizing hormone, is allegedly disclosed by Talwar, G. P., et al, Proc. Natl. 50 Acad. Set. U.S.A. 1976 73(1), 218—222. However, no disclosure of reducing and cleaving disulphide bonds is found in this article.

Bahl, O.P. et al, Biochem. Biophys. Res. Comm. 70, 525—532 (1976), disclose modifying the /5-subunit of human chorionic gonadotropin by reducing and cleaving disulphide bonds and alkylating the 55 reduced and cleaved disulphide bonds. These derivatives of hCG-/i have reduced immunological 55

reactivity with hCG, but show a greater loss in their immunological cross-reactivity with human luteinizing hormone. Those derivatives in which 3—5 of the total of 6 intrachain disulphide bonds are reductively cleaved and alkylated are reported to produce antibodies reactive with hCG. The derivative having all 6 disulphide bonds reductively cleaved and alkylated is reported to be immunologically 60 inactive. 60

This article also discloses that the specificity of hCG having 3 disulphide bonds and S-alkylated can be further enhanced by conjugation with tetanus toxoid. In contradiction to the above report of Talwar et al, this article reports that conjugation of the unmodified hCG-/5 with tetanus toxoid does not preferentially reduce its immunological cross-reactivity with human luteinizing hormone.

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GB 2 051 081 A 2

Therefore, a need'exists for a derivative of human chorionic gonadotropic which can act as an antigen to stimulate the formation of antibodies specifically to hCG with reduced or eliminated immunological cross-reactivity to hLH. Such a derivative would be useful both in preparing antisera for use in pregnancy testing by immunoassay and as a vaccine for preventing or terminating pregnancy.

It is therefore one object of this invention to provide a method of testing for pregnancy in humans. A further object is to provide a convenient and sensitive method of testing for pregnancy which can detect pregnancy soon after conception.

A further object is to provide a pregnancy test based on detecting the presence of human chorionic gonadotropin in body fluids such as serum and urine.

A further object is to provide antigens suitable for producing antisera capable of reacting specifically with human chorionic gonadotropin and having reduced cross-reactivity with human luteinizing hormone.

A still further object is to provide such antigens which produce antisera devoid of cross-reactivity with human luteinizing hormone.

A further object is to provide a method of contraception by administering an antigen capable of neutralizing the effect of human chorionic gonadotropin.

A further object is to provide such antigens by chemically modifying the /3-subunit of human chorionic gonadotropin.

It has now been discovered that antisera to human chorionic gonadotropin having reduced immunological cross-reactivity with human luteinizing hormone and useful in testing for pregnancy by immunoassay can be produced by using antigens comprising the /3-subunit of human chorionic gonadotropin which has been modified by reductively cleaving and alkylating all six of the intrachain disulphide bonds as well as 3, 4 or 5 of the bonds. Optionally, the hCG-/3 having three to six of its disulphide bonds cleaved and alkylated may be further conjugated with a protein to enhance its immunogenicity.

There have now been provided antisera, which are generally selective in reactivity to human chorionic gonadotropin to a distinguishing degree from luteinizing hormone, which are produced by the process comprising:

isolating the /3-subunit of human chorionic gonadotropin (hCG-/3);

cleaving from 3 to 6 of the intrachain disulphide bonds of the /3-subunit;

alkylating the thus reduced intrachain disulphide groups;

optionally conjugating the thus modified /3-subunit with a protein or hapten which functions to enhance the antibody response to the antigen when it is administered to a host animal;

administering the antigen so produced to a host animal whereby antibodies to the antigen are produced within the host animal; and isolating from the host animal an antiserum containing the antibodies to the administered antigen. This forms the subject matter of our copending British Patent Application No. 79.03645 (Case 5). Alternatively, the /3-subunit of hCG modified by oxidatively cleaving 3—6 of the intrachain disulphide bonds, optionally conjugated with a protein or hapten may be administered to the host animal to generate the antibodies which are subsequently recovered.

Such an antiserum is suitable for use in standard immunoassay methods for detecting the presence of human chorionic gonadotropin in body fluids such as blood and urine.

According to the present invention a process for producing an antigen which is capable of being administered to a host animal to induce an antibody response which is generally selective to human chorionic gonadotropin to a distinguishing degree from luteinizin hormone, comprises:

isolating the /3-subunit of human chorionic gonadotropin, reducing and cleaving three of the intrachain disulphide bonds of the said /3-subunit, alkylating the thus reduced intrachain disulphide groups, conjugating the said antigen with a protein or hapten comprising albumin, hemocyanin, thyroglobulin, a hapten or a peptide hapten effective to stimulate immune response,

either the said reducing, cleaving, and alkylating, or the said conjugating being carried out first, and isolating the antigen produced thereby.

The invention also comprises preventing or terminating pregnancy by administering to a woman an antigen prepared by:

isolating the /3-subunit of human chorionic gonadotropin (hCG-/3);

cleaving 3 of the intrachain disulphide bonds of the /3-subunit;

alkylating the thus reduced intrachain disulphide groups;

conjugating the thus modified /3-subunit with albumin, hemocyanin, thyroglobulin, a hapten or a peptide hapten which functions to enhance the antibody response to the antigen.

Prevention or termination of pregnancy can also be achieved by administering to the woman the antigen prepared by oxidatively cleaving the intrachain disulphide groups in hCG~/3, optionally conjugated with a protein or hapten.

Our copending British Patent Application No. (Case 5 Div 1) is directed to the invention which comprises an antigen comprising the /3-subunit of human chorionic gonadotropin which has from 4 to 6 of its disulphide bonds reductively cleaved and alkylated optionally conjugated with a protein or hapten to enhance its immunological specificity. Alternatively, the /3-subunit of hCGT may be modified

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GB 2 051 081 A 3

by oxidatively cleaving the intrachain disulphide linkages.

In practicing this invention human chorionic gonadotropin is prepared by any standard method. Material of a relatively low degree of purity is generally commercially available. The hCG is then purified by conventional techniques. A suitable procedure for this purification is that of Bahl, O.P., J. Biol. Chem.

5 244:567,1969. 5

The following is illustrative of the purification technique for providing the hCG:

The purification procedure involves three steps of column chromatography carried out at 4°C. The crude hCG, obtained commercially, is subjected to chromatography on a column of DEAE-sephadex A-50 previously equilibrated in 0.04 M tris-phosphate buffer, of pH 7.5. The elution of the column is 10 initiated with the above equilibrating buffer followed by a series of buffers of increasing NaCI 10

concentration in a discontinuous step-wise gradient. Each function from the column is monitored for protein by measuring absorbance at 278 nm and for the hormonal potency by a radioimmuno-assay. The most active fraction is then subjected to another column of DEAE-Sephadex A-50 equilibrated with 0.04 M tris-phosphate buffer of pH 7.5 containing 0.1 M sodium chloride. The column is then eluted 15 with a continuous gradient between 0.1 M sodium chloride and 0.2 M sodium chloride in 0.04 M tris- 15 phosphate buffer of pH 7.5. Finally, the major hormone fraction from the previous step is purified by chromatography on a column of Sephadex G-100 equilibrated with 0.04 M sodium phosphate buffer of pH 7.5. The column is eluted with the same buffer. The active fraction is concentrated by an Amicon ultrafiltration ceil, dialyzed and lyophilized.

20 The /3-subunit of hCG is then isolated by dissociating the purified hCG followed by successive 20 chromatography. It is desirable to use a stepwise rather than a continuous gradient chromatography.

This procedure is described in Swaminathan, N., and Bahl, O.P., Biochem. Biophys. Res. Comm. 40:422, 1970; Bahl, O.P. in Hormonal Proteins and Peptides, C. H. Li. Ed., Acad. Press, p. 170, 1973.

Conventional ion-exchange columns for isolating protein fractions may be used, these include 25 carboxymethyl cellulose, phosphocellulose, carboxymethyl sephadex, sulpho-propyl sephadex, and 25 hydroxy appatite. For best results, a step-wise elution is used. That is, the salt concentration in the buffer solution used to elute the hCG-/3 is increased from 0.0 M up to about 3 or 4 molar in a step-wise fashion.

The following is illustrative of a technique for isolating the /3-subunit:

30 The purification of hCG-/3 involves the dissociation of hCG in 0.1 M sodium acetate buffer of pH 30 5.5 in the presence of 8 M ultrapure urea at 40°C for 1 hour followed by the separation of the dissociated subunits on a column of DEAE-Sephadex. Before the application of the sample, the column is equilibrated with 0.04 M tris-phosphate buffer of pH 7.4. The elution of the column is initiated with the equilibrating buffer and continued by discontinuous step-wise gradient with buffers of increasing 35 salt concentration ranging from 0.05 M to 0.3 M in 0.04 M tris-phosphate buffer of pH 7.4. The a- 35 subunit is eluted with the initial buffer while the /3-subunit is eluted from the column with the buffer containing 0.1 to 0.2 M sodium chloride. The /3-subunit fraction is further purified by chromatography on a column of Sephadex G-100, previously equilibrated and eluted with 0.1 M sodium acetate buffer of pH 5.0. All operations of column chromatography are preferably carried out at 4°C. In order to remove 40 the last traces of contaminating hCG-ar or hCG from the /3-subunit, it is passed through a column of an 40 immuno-adsorbent prepared by treating a pooled hyperimmune rabbit anti hCG-ar serum with glutaraldehyde according to the standard procedure.

In order to ensure removal of undissociated hCG and of the a-subunit of hCG (hCG-or), the purified hCG~/3 may be treated with anti-hCG-a immuno-adsorbent.

45 Techniques for preparing anti-hCG-a: immuno-adsorbents are well known. A technique for 45

preparing such adsorbents is taught by Avrameas et al, Immuno-chemistry, 6:53 (1969).

The protein modification by the clearance of disulphide bonds either by oxidation or by reduction to produce —S03H or two —SH groups in place of one disulphide linkage is a conventional procedure although not previously applied to the /3-hCG. Commonly used oxidizing agents include performic acid, 50 halogen, hydrogen peroxide, air oxidation and sodium sulphide. The oxidation conditions are those 50 conventionally utilized for cleavage of disulphide linkages. Thus, the oxidation may be performed in an aqueous medium at temperatures of from 0—40°C, though lower or higher temperatures may be used if desired. The oxidation process oxidizes the intra-chain disulphide groups in the cystine residues and the sulphide groups in the methionine residues of the hCG-/3. However, for the preparation of the 55 specific antigen, reduction and alkylation is preferred to oxidation. 55

Usable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), /3-mercaptoethanol and sodium borohydride. DTT and DTE are the preferred reducing agents. The number of disulphide bonds reduced and'cleaved is controlled by adjusting the reaction conditions, particularly the amount of reducing agent employed, the concentration of protein in the reaction mixture, the reaction time, 60 temperature and pH. Preferably, the reduction is carried out under mild conditions so that the intrachain 60 disulphide linkages in the cystine residue are selectively reduced and to minimize reduction of the other amino acid residues of the protein, i.e., the tyrosyl, trysyl, hiptidyl and methionine residues. This can be readily accomplished by control of temperature, pH or reaction time.

The reduction can be carried out at room temperature or at temperatures slightly above or below 65 room temperature, i.e. between 0°C and 40°C. 65

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GB 2 051 081 A 4

Preferably the reaction is conducted at about 37°C.

The reduction is conveniently conducted at a mildly alkaline pH, e.g. about 7.2—9, preferably about 8—8.6.

The reducing agent can be added as a solution to the protein solution, or vice versa, or as a solid to the protein solution. Alternatively, and less desirably, the protein can be dissolved in the reducing 5

solution or the reducing agent and the protein can be dry mixed and then dissolved simultaneously in the aqueous reaction solvent to the desired protein concentrations, e.g. at least about 1 per cent,

preferably about 5—18 per cent.

The extent of the reaction can be followed, if desired, by monitoring the reaction mixture for residual reducing agent. When reducing agent is no longer being consumed, the reaction is complete. 10 „

The reaction can be conducted in air or under an inert atmosphere, e.g. of nitrogen. It is preferred to conduct the reduction under a nitrogen atmosphere.

The molar concentration of reducing agent to protein is dependent in part on the protein concentration in the reaction mixture, pH, the reaction time, the reaction temperature, and reaction atmosphere. At least one molar equivalent of reducing agent is theoretically required to reduce one 15 disulphide linkage per mole of protein and produce 2 —SH groups. Therefore, the theoretical minimum amount of reducing agent required is at least one molar equivalent for each disulphide linkage to be reduced. Hence, at least about 3 molar equivalents of reducing agent must be employed in producing the modified hCG-/3 of this invention. It is preferred to employ an excess of reducing agent ranging from 2 to 20 times the theoretical amount to reduce and cleave the desired number of disulphide linkages. 20

The protein is preferably present in a concentration of about 4 x 10-s molar to 8 x 10-s molar.

Hence, the reducing agent, when the preferred reducing agents dithiothreitol and dithioerythitol are used, is preferably present at concentrations from about 5 x 10-4 molar to about 9 x 10""3 molar.

In the course of experimentally determining the conditions required for reducing and cleaving a particular number of disulphide bonds, the number of disulphide bonds reduced and cleaved in a 25

particular experiment is determined by amino acid analysis of the modified protein. This analysis is carried out by standard procedures well known to those skilled in the art. Each cleaved disulphide linkage will yield two cysteine (half cystine) residues. The number of disulphide bonds cleaved in a given experiment is therefore equal to one-half the number of moles of cysteine found divided by the total number of moles of protein. 30

The sulphhydryl groups produced by the reduction must be protected, otherwise the disulphide linkage may reform. The necessary protection can be achieved by simply alkylating the sulphhydryl groups with an appropriate alkylating agent. The alkylating agent may be any of those conventionally employed provided the resulting mercapto group is stable and physiologically acceptable.

For purposes of this application, the term "alkylating" as applied to the sulphhydryl groups formed 35 by reductive cleavage of the disulophide groups means replacing the hydrogen atom of the sulphhydryl group by a carboxymethyl, carboxyamidomethyl, aminoethyl, or equivalent group. This procedure is also a frequent procedure in protein chemistry and is carried out by well known methods.

The alkylation is conveniently carried out in the same reaction vessel as the reductive cleavage.

Sufficient alkylating agent is employed to react with any residual reducing agent and to convert all of 40 the free —SH groups present in the reduced product to alkylated mercapto groups. The exact nature of the alkylating agent is not critical, provided that the resulting alkylated mercapto group is stable and physiologically acceptable.

In a preferred embodiment, the alkylated mercapto groups have the formula —S—CH2—R wherein R is —H, —CH3, — COOH, — COOR', —C0NH2, —CONHR', — C0N(R')2, — C=N, —-CH2Ce=N, 45 CH2NH2, —COPh, —CH2OH, or

—CH—OH,

I

CH3

wherein R' is lower alkyl, i.e. containing 1—4 carbon atoms, and Ph is unsubstituted phenyl or phenyl bearing 1—3 single substituents such as, e.g. lower alkyl, chloro, bromo, nitro, amido, lower alkoxy or lower alkoxycarbonyl. Examples of such substituted phenyl groups include p-tolyl, sym-xylyl, p- 50

amidinophenyl, m-chlorophenyl and p-methoxyphenyl. The alkylated mercapto group can also be

—S—CH—CH2—CON(R')—CO,

1 —J

S—(lower alkoxycarbonyDR', e.g. ethoxycarbonylethylmercapto, and —S—(carboxy)—R\ e.g. ethoxycarbonylmercapto, or other lower alkylmercapto groups having a functional group on the carbon atom of the alkyl group. 55

The alkylation conditions are substantially the same as those employed in the reduction step.

Somewhat longer reaction times, e.g. 1—2 hours may be employed.

The alkylation of the—SH groups produced by reductive cleavage of the disulphide linkages is

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GB 2 051 081 A 5

preferably accomplished by using iodoacetamide or iodoacetic acid to produce pairs of—SCH2CONH2 or —SCH2COOH groups, respectively. However, the —SH groups also can be blocked by alkylating the reduced hCG-/3 with other alkylating agents to produce a modified HCG-/3 having substantially the same physical and biological properties as that in which the reduced cleaved intrachain disulphide linkages 5 are replaced by pairs of—SCH2CONH2 groups. 5

Alkylation of the —SH groups to give —S—CH2—R groups wherein R is hydrogen or methyl can be accomplished by treating the reduced hCG-/3 with, for example, methyl and ethyl iodide, respectively. Substituted alkylthioethers can also be formed by using a haloacetamiae such as iodoacetamide, N-alkyl-haloacetamide or N,N-dialkyl-haloacetamide, e.g. BrCH,CONHC3H7 or BrCH2CON(C2H5); haloacetic 10 acid or its lower alkyl esters, e.g. iodoacetic acid, ICH2C02C2H6 or CICH2C02C2Hs; haioacetonitrile, e.g., 10 ICH2CN; alkenylnitride, e.g. acrylonitri'e; aralkyl halide, e.g. benzyl bromide; alkylene oxide, e.g. ethylene oxide: phenacyl halide, e.g. phenacyl chloride and phenacyl bromide; N-alkylmaleimide, e.g. N~ ethylmaleimide; a-halo lower alkanoic acids of 3 or 4 carbon atoms and lower alkyl esters thereof, e.g.

ethyl a-bromopropionate and a-bromopropionic, and alkylene imines, e.g. ethyleneimine. The alkylation 15 reactions are conducted under mildly alkaline conditions. 15>

The reaction conditions employed are those conventionally used with the selected alkylating agent. With the very reactive agents, between about 5 and 10 molar equivalents are generally employed, the exact amount depending in part on the reaction temperature and time, concentration of the protein and alkylating agent in the solution, and the number of free —SH groups per molecule. With 20 less reactive agents, e.g. ethylene oxide, a large molar excess may be required to achieve the desired 20 complete alkylation of all free —SH groups.

To fully alkylate the product of the reduction step, at least two molar equivalents, calculated on the equivalents of reducing agent used in the reduction step usually are required. Preferably, about a 10 per cent or greater molar excess is employed to ensure complete alkylation of all residual reducing 25 agent and the conversion of all protein —SH groups to —S—CH2—R groups. For example, if DTE is 25 employed at 8.4 x 10~4 molarity initial concentration in the reaction mixture, the preferred initial molarity of the alkylating agent in the reaction mixture is about 1.7 x 10~3. When employing a gaseous alkylating agent, e.g. ethylene oxide, a larger molar excess, e.g. 10 to 50 molar equivalents or more may be required to alkylate completely all free —SH groups. Larger molar excesses can be employed, e.g. up 30 to 20 molar equivalents, without detrimental results, since the excess alkylating agent is later separated 30 from the alkylated product after the alkylation, e.g. by precipitating the protein, dialysis, or desalting on a Sephadex column.

It is preferred to alkylate with iodoacetamide, iodoacetic acid, or ethyleneimine. When alkylating with iodoacetamide, it is preferred to use a 4 to 25 fold excess or reagent. The reaction is preferably run 35 at about 37°C and a pH of about 8.5 for a period of about 30 minutes. Whan alkylating with iodacetic 35 acid or ethyleneimine it is preferred to use a 50-fold excess of reagent under the above conditions. It is preferred to use a buffer such as 0.5 M tns hydrochloric acid buffer to maintain the desired pH. It is preferred to use a buffer containing 8 M urea and 2% ethylenediaminetetraacetic acid groups.

Conjugation or cross linking with a protein may be carried out by any standard procedure such as 40 by reacting the hCG-/3 or its derivatives and the protein in aqueous solution with glutaraldehyde 40

according to Avrameas, S., fmmunochemistry, 6:53, 1969 or with a water soluble carbodiimide according to Cutarcasas, P. and Anfinsen, C.B., Methods of Enzymology, XXII: 343, 1971. Other procedures using reagents such as ethylchloroformate, bifunctional arylhalides, such as 1,3 or 1,4 difluoro- or dischloro-benzene, 2,4-difluoro- or dichloro-toluene, 4,4-difluoro- or dichloro-bi-phenyl and the 45 jike, 1,5-difluoro-2,4-dinitrobenzene, bifunctional isocyanates, such as toluene 2,4-diisocyanate, toluene 2,6- 45 diisocyanate, 4,41-diisocyanatodiphenyl methane, or hexane 1,6-diisocyanate and bifunctional acylating agents such as di-acid halide, carboxylic dianhydrides, dicarboxylic acids, and esters and diamides, and imiidoesters may also be used.

Conjugation procedures using glutaraldehyde or a water soluble carbodiimide, are preferred. 50 In the preferred conjugation procedure using glutaraldehyde, the modified hCG-/5 or its derivatives 50

and the protein to be conjugated are mixed in an aqueous phosphate buffer (pH about 6.8). An aqueous glutaraldehyde solution (about 1% glutaraldehyde) is added slowly, with stirring and the mixture is allowed to stand at room temperature for about 3 hours. The solution is then dialyzed, polymerized material is removed by centrifuging (e.g. 50,000 g for 30 minutes), and the supernatant liquid is 55 subjected to column chromatography (e.g. on Sephadex G-150) to separate the free hCG-/3 or its 55

derivatives from the conjugate.

In the carbodiimide procedure the hCG-/3 or its modified form and the protein in aqueous solution at pH 4.75, are treated with a five fold excess of the carbodiimide. The pH of the reaction is maintained by the addition of 0.001 N hydrochloric acid. The reaction is carried out at 25°C for 1 hour following 60 which the reaction mixture is dialyzed against water and lyophilized. 60

Conjugation with a hapten such as adjuvant peptide, (muramyl alanyl isoglutamine) is carried out as follows. The peptide is activated with the carbodiimide at pH 4.75 at 25°C for 15 minutes. Then the hCG-fi or its derivative is added and the reaction is allowed to proceed for 1 hour at 25°C. The pH of the reaction mixture is maintained at 4.75 with 0.001 N hydrochloric acid. The reaction mixture is then 65 dialyzed and lyophilized. 65

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The albumin which is conjugated with the hCG-/3 or its derivatives include serum albumin derived from bovine, rabbit, or human sources, oval albumin, methylated albumin, that is essentially any albumin. Conjugation is accomplished using the procedure outlined previously for conjugating the hCG-

/*•

The haptens which are conjugated with the hCG-/5 or its derivatives are essentially any compound 5 capable of modifying the functional groups on the protein, particularly the amino groups. The presence of hapten on the peptide enhances the immunological specificity of hCG-/3. The haptens may also function as a protecting group for the amino portion of the protein. Suitable haptens include conventional mono-functional alkylating and acylating agents. The alkylating agents which are suitable include those utilized to alkylate the sulphhydryl linkages as well as compounds such as 2,4- 10

dinitrofluorobenzene, chloro orfluorobenzene, chloro or fluorotoluene, that is, arylhalides and alkyl halides. Acylating agents include acid anhydrides such as acetic anhydride, succinic anhydride, N-carboxy-a-amino anhydrides, O-carbobenzoxy tyrosyl-amino-anhydride, or O-carbobenzoxy-L-tyrosin-N-carboxy-ar-amino anhydride, acid halides, carboxylic acids and their esters, and amides.

An example of the peptide haptens which stimulate immune response and which in accordance 15 with the invention are conjugated with the hCG-/5 is muramyl alanyl isoglutamine. With peptide haptens it is necessary to employ one of the conventional conjugating agents, such as those described previously, to bind the peptide hapten to the hCG-/5 or its derivative. The carbodiimide, described previously, is preferred. Obviously, no conjugating agent is required when the hapten contains a functional group capable of directly reacting with hCG-/} or its derivative. 20

When the hCG-/3 or its derivative is modified with a hapten from 1—10 hapten molecules per mole of hCG-/3 or its derivative is present on the protein; preferably from 1—5 hapten molecules, most preferably 2—3 hapten molecules per mole of protein are present. When the hCG-/3 or its derivative is to be used either as a contraceptive vaccine or to induce abortion it is preferable to limit the number of tyrosyl residues incorporated on the protein. Preferably no more than two tyrosyl residues are present in 25 the hCG-/3 or its derivative. When the hCG-/3 or its derivative is to be used to produce an anti-serum,

such as that used for pregnancy testing, the number of tyrosyl units is not critical and may range up to ten.

It is preferred to protect the amino groups by attaching O-carbobenzoxytyrosyl residues. This is preferably accomplished by reacting hCG-/3 or a derivative in an aqueous 0.05 M phosphate buffer of pH 30 about 7.2, at a concentration of about 6.6 x 10~s molar with a 50 fold excess of O-carbobenzoxy-L-tyrosine-N-carboxy-ar-amino anhydride dissolved in dioxane at a concentration of about 5.0 x 10-2 molar. The reaction is preferably carried out at 4°C for 24 hours, after which the O-carbobenzoxytyrosyl derivative may be isolated by desalting on a Sephadex column (e.g. G-25).

hCG-/3 or its derivative may also be modified by coupling the carboxyl groups with a nucleophile, in 35 particular, those nucleopiles capable of blocking the carboxyl group with an amino group. Suitable nucleophiles include amino acids and their esters, small peptides, and proteins, such as glycine ethyl ester, tyrosine ethyl ester, alanyl ethyl ester, glutamyl diethyl ester, aspartyl diethyl ester, lysine ethyl ester, and histadine ethyl ester. That is, essentially any compound containing an amino group may be used. The coupling can be carried out by reacting an aqueous solution of hCG-/3 or a derivative with a 40 nucleophile, e.g. glycine ethyl ester hydrochloride in the presence of urea. The reaction is initiated by the addition of a carbodiimide e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The reaction is carried out at an acidic pH, e.g. about 4—6 and at from 0—50°C. The reaction medium is not critical. After the reaction has proceeded to some extent, more carbodiimide may be added to bring the reaction to completion. 45

Preferred nucleophiles are glycine ethyl ester and tyrosine ethyl ester. It is preferred to couple these nucleophiles to the carboxyl groups of hCG-/5 or its derivatives by adding to an aqueous solution of hCG-ji or derivative at a concentration of about 6 x 10-5 molar, a one molar solution of the nucleophile as the hydrochloride salt and a 7.5 molar solution of urea. The reaction is initiated by adding 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to a concentration of about 0.07 molar. 50 The pH is maintained at about 4.75 with 0.1 N aqueous hydrochloric acid solution during the reaction.

After about 60 minutes an equal amount of the carbodiimide is added to bring the final concentration to about 0.14 molar. The reaction is continued for about another 60 minutes, and then solid ammonium carbonate is added to stop the reaction.

The antigen prepared by the above procedures may then be used to produce antibodies to hCG 55 either in animals, whereby an antiserum useful in testing for pregnancy is produced or in humans,

whereby pregnancy is prevented or terminated.

The antiserum is prepared by conventional procedures utilized in the preparation of other types of antibody serum. That is, a host animal such as a horse, goat, sheep, rabbit, monkey or pig is injected with antigen on a regular basis until their blood contains the desired level of antiserum. The injection 60 schedule for the antigen is not critical, it may be injected as often as practical. In practice, injection every other week has proved satisfactory. Longer or shorter periods between injection are, of course, possible. The dosage of antigen is, of course, proportionate to the weight of the host animal. The minimum dosage is that required to induce an antibody response in the host, while the maximum is that at which no adverse side reactions occur. Generally, dosages from about 2 /ug/kg to about 50 fig/kg of 65

7

GB 2 051 081 A 7

body weight will prove satisfactory; preferably from about 5—50 fig/kg of body weight.

Further details of the method of preparation and use of the antisera are given in our copending British Patent Application No. 79.03645 referred to above.

In using the antigens of this invention to prevent or terminate pregnancy, the antigens are 5 administered to a woman to provoke the formation of antibodies which will neutralize hCG. An effective amount of the antigen is administered to the recipient by injection, preferably by the intravenous route. The total dosage unit which is to be administered in one or more injections is determined by the body weight of the recipient. The total dose will, in general, be several micrograms per kilogram or less. That is, the quantity of antigen administered is sufficient to induce the required antobody response but 10 insufficient to induce an adverse reaction. The dosage will normally be from about 2—50 /ug/kg of body weight, preferably from 2—10 /ug/kg of body weight. Thus, the typical dosage will range from 100—500 ^g. Generally, the dosage will be repeated several times to stimulate the necessary response usually from 2 to 4 times. The antigen may be administered in the form of solution in a human adjuvant such as are commercially available. One such adjuvant is an isotonic sodium chloride solution. 15 It is also possible to administer the antigen orally by combining the antigen with lipids. In this manner, it is absorbed from the intestines directly into the blood.

The procedure to either terminate pregnancy or prevent conception are essentially identical. The antigen in the carrier—the vaccine—is administered in accordance with the procedure outlined previously. When it is desired to prevent conception, the antigen will be periodically administered to 20 maintain the necessary antibody level in the woman.

In a preferred embodiment the invention comprises an antigen which is capable, when administered to a host animal, of inducing an antibody which is generally selective to human chorionic gonadotropin to a distinguishing degree from luteinizing hormone which comprises a /3-subunit of human chorionic gonadotropin which has been reduced such that three of the intrachain disulphide 25 bonds are cleaved, the cleaved disulphide bonds alkylated, and the subunit further conjugated with albumin, hemocyanin, thyroglobulin, or muramyl alanyl isoglutamine which function to enhance the antibody response to the antigen when the antigen is administered to the host animal.

Having generally described the invention, a more complete understanding can be obtained by reference to certain specific examples, which are included for purposes of illustration only and are not 30 intended to be limiting unless otherwise specified.

In the following Examples, DEAE-Sephadex is a commercially available chromatographic packing material comprising diethylaminoethyl Sephadex and Sephadex G-100 is a cross-linked polydextran packing material.

EXAMPLE 1

35 This Example illustrates the preparation of the precursor antigen for the use in producing conjugated antigens according to this invention, which are shown in Examples 2 and 3.

Preparation of hCG and its Subunits.

Human chorionic gonadotropin (12,000 lu/mg) was prepared from a commercial preparation having a potency of 4,100 lu/mg essentially by the purification procedure of Bahl, O.P., J. Biol. Chem. 40 244:567,1969, except that a pH of 7.4 was maintained throughout the purification.

The /3-subunit of hCG was prepared by dissociation of hCG with 10 M urea followed by chromatography successively on columns of crosslinked polysaccharide (DEAE-Sephadex) using a stepwise rather than a continuous gradient and Sephadex G-100, following the procedure of Swaminathan, N. and Bahl, O.P., Biochem. Biophys. Res. Comm. 40:422, 1970.

45 Treatment ofhCG-fi with Anti-hCG-a Immunoadsorbents.

In order to ensure the removal of trace amounts of hCG-a or hCG, if present, hCG-/3 was treated with anti-hCG-ar immunoadsorbent. The immunoadsorbent was prepared from pooled sera of anti-hCG-a by the method of Avrameas, S. and Ternynck, T., Immunochemistry 6:53, 1969, and tested for efficacy by the method of Bahl, et al., Biochem. Biophys. Res. Comm. 70:525, 1976. Two mg of highly 50 purified hCG~p> in 5 ml of phosphate buffered saline (PBS) was treated with 10 g (wet wt.) of immunoadsorbent for 2 hours at 25°C. The adsorbent was centrifuged and the sediment was washed with 10 ml of PBS. The supernatant liquid was concentrated by lyophilization and desalted on Sephadex G-25.

Radioligand Receptor Assay.

55 hCG-/3 preparations were evaluated for any hCG contamination by radioligand receptor assay

(RRA). The assay was carried out using rat testes homongenate as described in Bellisario, R. and Bahl. O.P. J. Biol. Chem. 250:3837, 1975.

5

10

15

20

25

30

35

40

45

50

55

Modification of Disulphide Bonds by Graded Reduction ofhCG-[i and Carboxyamidomethylation

To 0.2 /imol of hCG-/3 in 3.5 ml. of 0.5 M tris hydrochloric acid buffer of pH 8.5, containing 8 M 60 urea and 2% ethylenediaminetetraacetic acid, was added 2 to 25 /imol of dithioerythritol. The reaction 60

8

GB 2 051 081 A 8

mixture was incubated at 37°C for 30 minutes under nitrogen as described in Carlen, R. B. Bahl, O.P., and Swaminathan, W., J. Biol, Chem. 248:6810,1973. After addition of 5 to 60 jumol of iodoacetamide the incubation was continued for another 30 minutes. The reduced and S-carboxyamidomethyl derivatives of hCG-/3 were desalted on a coarse Sephadex G-25 column (2.5 x 100 cm) and the 5 fractions under the protein peak were pooled and lyophilized. Table 1 illustrates the experimental conditions for producing the various degrees of bond cleavage and alkylation.

Modification of Disulphide Bonds by Reduction and S-Carboxymethylation or S-Aminoethylation of hCG-[i.

The reduction of the disulphide bonds in hCG-/3 was carried out as described above. The 10 sulphhydryl groups thus formed were reacted with a 50-fold excess of iodiacetic acid for S- 10

carboxymethylation or ethyeneimine for S-aminoethylation by the procedure of Carlsen, et al. The derivatives were then desalted on a Sephadex G-25 column.

TABLE 1

Conditions for Reduction and S-Carboxyamidomethylation

Amount of hCG-/3 (jumol)

Volume of reaction mixture (ml)

Amount of diethioerythritol (jumol)

Amount of iodoacetamide (jumol)

Number of1 S-carboxyamidomethyl cysteine residues

0.09

2

1.5

3.0

4.6

0.09

2

1.0

2.4

6.3

0.22

5

2.5

7.5

4.9

0.35

5

4.2

8.4

0.35

5

21.8

48.6

9.4

0.35

5

42.3

83.8

12.2

determined by amino acid analyses.

Protection of Amino Groups by O-Carbobenzoxytyrosyl Residues.

15 To 1.5 ml of a solution of 0.1 ^itmol of DS3-hCG-/3 in 0.05 M sodium phosphate buffer of pH 7.2, 15 was added 5 jumol of O-CBZ-L-tyrosine N-carboxy-a-amino anhydride dissolved in 0.1 ml of dioxane. The reaction was allowed to proceed at 4°C for 24 hours after which time the derivative, 0-CBZ-Tyr-hCG-/5, was isolated by desalting the mixture on a Sephadex G-25 column.

This procedure was also used to prepare DS3-Tyr5-hCG-/3.

20 EXAMPLES 2A, 2B AND 2C

Conjugation ofhCG-fi or its Derivatives with Rabbit Albumin Using Glutaraldehyde.

To 5.8 mg of hCG-/3 or its derivatives (prepared as in Example 1) 10 mg of rabbit albumin in 1.0 ml of 0.1 M sodium phosphate buffer of pH 6.8 was added 100 mi of 1% glutaraldehyde in small instalments. The reaction mixture was kept at room temperature for 3 hours. Any insoluble polymerized 25 material is removed by centrifuging at 15,000 rpm for 15 minutes. The unreacted glutaraldehyde and salts were removed by chromatography on a column of Sephadex G-25. A similar procedure may be followed for other proteins such as hemocyanin (Example 2B) and thyroglobulin (Example 2C).

EXAMPLE 3

Conjugation ofhCG-ji or Its Derivatives with Hemocyanin Using Carbodiimide Procedure.

30 5 mg of hCG-/3 or its derivatives (prepared as in Example 1), 10 mg of hemocyanin and 30

carbodiimide (1 fi mole) were dissolved in 1 ml of water. The pH of the resulting solution was adjusted to pH 4.75 with 0.001 N HCI. The reaction was carried out for 60 minutes at room temperature. Hemocyanin-hCG-/3 complex was separated from free hCG-/3 on a column of Sephadex G-100 which was eluted with 0.1 M ammonium bicarbonate.

35 Modification of the Carboxyl Groups of hCG-fi by Coupling with Glycine and Tyrosine Ethyl Esters. 35

To 3.5 ml of an aqueous solution of hCG-/3 (0.2 ^mol) was added glycine ethyl ester hydrochloride ortyrosin ethyl ester hydrochloride (1 M) and urea (7.5 M). The reaction was initiated by the addition of

20

25

9

GB 2 051 081 A 9

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) ot a concentration of 0.07 M.The pH was maintained at 4.75 with 0.1 N hydrochloric acid during the reaction. After 60 minutes, an equal amount of the carbodiimide was added to bring the final concentration to 0.14 M. The reaction was continued for another 60 minutes, and then solid ammonium carbonate was added to terminate the 5 reaction. 5

Degree of Modification by Amino Acid Analysis.

In order to determine the extent of modification of hCG-/5 derivatives (50—150 /ug) were hydrolyzed for amino acid analyses with 0.5—1 ml of 5.7 N hydrochloric acid in evacuated sealed tubes at 110°C for 24 hours. After removal of the acid by a rotary evaporator, the hydrolysate was analysed 10 on a Spinco Model 120C Automatic Amino Acid Analyzer equipped with a range expander on the 10

recorder. The protein content of the samples was calculated from the amounts of aspartic acid or glutamic acid obtained in the amino acid analysis and from the number of residues of each as determined from the amino acid sequence of hCG-/5.

Multiple Modifications ofhCG-[5.

15 hCG-/5 was first modified by any one of the above methods. The resulting singly-modified hCG-/5 15 derivatives were further modified by using other blocking agents under the appropriate conditions described above. The derivatives resulting from double modifications were then conjugated with tetanus toxoid or other protein using glutaraldehyde. All derivatives were characterized at each stage by amino acid analyses.

20 EXAMPLE 4 20

This Example illustrates the evaluation of the immunological activity of the modified /5-subunit of human chorionic gonadotropin.

Preparation of Antisera.

Antisera to hCG-a, hCG-/5, hCG, and hLH were raised in New Zealand male rabbits. A 25 hyperimmune schedule was followed to obtain high titer and high-affinity antibody as disclosed by 25 Avrameas and Ternynck. For RIA, pooled antisera from more than one rabbit was used. The antibody titer was measured by using 12Sl-labelled hormones.

Radioimmunoassays for hCG, hCG-p and hLH.

hCG, hCG-/3 and its analogues for hLH were iodinated with Na125l using the chloramine-T 30 procedure described in Bellisario, et al. The radioimmunoassays (RIA) were carried out as described in 30 Tomada, Y. and Hreshchyshyn, M. M.,Amer. J. Obst. Gynae., 100:118,1962, except that the incubation time was either 16 hours at 2b°C or 2 hours at 37°C, followed by 16 hours at 4°C. The free and the antibody-bound hormones were separated by ethanolic ammonium acetate or by the double antibody technique using sheep anti-rabbit ^-globulin as described in Vaitukaitis, J. L., et al..Amer. J. 35 Obst. Gynae. 113:751,1972. The relative activities of the hCG-/3 and its derivatives were determined in 35 the hCG-/3-anti-hCG-/3 system (hCG-/3 system) by measuring their ability to displace 50% of 12Bl-hCG-/5. In some cases, the hCG-anti-hCG system was also used. To determine the cross-reactivity of the hCG-6 derivatives with hLH, the 1z5l-hLH-anti-hLH system (hLH system) was used.

The results of the evaluation of the effect of reduction and alkylation of the disulphide bonds are 40 shown in Table 2. In Table 2, the particular modifications of hCG are indicated by the prefixed. Thus, 40 DS3— indicates that three disulphide bonds have been cleaved and alkylated with carboxyamidomethyl groups.

TABLE 2

Effect of Reduction and Alkylation of Disulphide Bonds on the Immunological Activity of hCG-/5 Number of hCG-/3/ disulphide Alkylating Percent Activity Ratio

Example Derivative bonds cleaved Reagent hCG-/5 System hLH System hCG-/5/hLH

1 hCG-/3 0.0 100 100 (4.5)2 1.0

2 DS3-hCG-/51 2.9 Iodoacetamide 35.0 1.8 16 0.5(0.72) 40.0

1The figures in subscripts represent the number of disulphide bonds modified; calculated from S-carboxymethylcysteine, S-aminoethylcysteine and cysteic acid residues obtained on amino acid analysis. 2The figures in parenthesis represent the actual values of LH activity of hCG-/5 and its derivatives in a 125lhLH-anti hLH system wherein hLH in this system is assumed to have activity of 100.

GB 2 051 081 A 10

Table 3 shows the effects of multiple modifications on the immunological activity of hCG-/3. In this table the prefixes have the same meaning as in Table 2 and Tyr signifies an O-carbobenzoxytyrosyl substituent. Gee signifies a glycine ethyl ester group.

TABLE 3

Effect of Multiple Modifications on the Immunological Activity of hCG-/3

Example hCG-/3/ Derivative

Number of disulphide bonds cleaved

Number of amino or carbonyl groups modified

Percent Activity hCG-/3 system hLH system

Ratio hCG-/3/ hLH

—NH z -

-COOH

3

hCG-/3

0

100

100 (4.5)2

1

4

DS3Tyrs-hCG-/31

2.9

5.2

4.50

0.10(0.004)

45

5

DS3TyrsTTrhCG-/3

2.9

5.2

1.0

1.80+0.1

0

OO

6

Gee2o-DS3-TT1-hCG-/3

3.1

1.0

20.0

1.60+0.1

0.80 (0.003)

20

7

DS3-Ty rg-TT, hCG-/3

3.1

5.2

32.0

CO

8

Gee20-DS3TT1hCG-/3

3.1

20.0

32.0

7.0

4.6

1The derivatives are abbreviated according to the order of modification. Figures in the subscripts indicate the number of groups modified or conjugated.

2The figures in parenthesis have the same significance as in Table 2.

Claims

5 CLAIMS 5

1. A process for producing an antigen which is capable of being administered to a host animal to induce an antibody response which is generally selective to human chorionic gonadotropin to a distinguishing degree from luteinizing hormone, comprising:

isolating the /3-subunit of human chorionic gonadotropin, reducing and cleaving three of the

10 intrachain disulphide bonds of said /3-subunit, alkylating the thus reduced intrachain disulphide groups, 10 conjugating the said antigen with a protein or hapten comprising albumin, hemocyanin, thyroglobulin, a hapten or a peptide hapten effective to stimulate immune response,

15

2. A process as claimed in Claim 1 in which the said reducing, cleaving, and alkylating is carried 15 out first and then the conjugating is carried out.

3. A process as claimed in Claim 1 or Claim 2 in which the said antigen is further modified by reaction of the terminal carboxy group thereof with a nucleophile.

4. A process as claimed in Claim 3 in which the said nucleophile is tyrosine ethyl ester or glycine

20 ethyl ester. 20

5. A process as claimed in any one of Claims 1 to 4 in which the said /3-subunit is isolated by dissociating human chorionic gonadotropin into a- and /3-subunits with a 10 Molar aqueous urea solution,

subjecting the said dissociated human chorionic gonadotropin to chromatography successively on

25 columns of a cross-linked modified polysaccharide using a step-wise gradient, and cross-linked 25

polydextran, whereby said /3-subunit is isolated.

and subsequently treating the isolated /3-subunit with an immunoadsorbent capable of adsorbing the said ar-subunit whereby any a-subunit present is removed.

6. A process as claimed in Claim 1 substantially as specifically described herein with reference to

30 any one of the accompanying Examples 2A, 2B, 2C, 3,4.2 or 4.4 to 4.8. 30

7. An antigen which is capable of being administered to a host animal to induce an antibody response which is generally selective to human chorionic gonadotropin to a distinguishing degree from luteinizing hormone, which comprises a /3-subunit of human chorionic gonadotropin which has been reduced such that three of the intrachain disulphide bonds have been cleaved, and wherein the said

35 cleaved disulphide bonds have been alkylated, and wherein the said subunit is further conjugated with a 35 protein or hapten comprising albumin, hemocyanin, thyroglobulin, a hapten or a peptide hapten effective to stimulate immune response, which enhances the immunogenetic speficity of the said antigen.

11

GB 2 051 081 A 11

8. An antigen which is capable of being administered to a host animal to induce an antibody response which comprises a /5-subunit of human chorionic gonadotropin which has been oxidized such that three of the intrachain disulphide bonds have been oxidatively cleaved, and in which the said /}-subunit after said oxidative cleavage has been conjugated with a protein or a hapten comprising

5 albumin, hemocyanin, thyroglobulin or muramyl alanyl isoglutamine, which functions to enhance the 5 antibody response to said antigen when said antigen is administered to said host animal.

9. An antigen as claimed in Claim 8 in which the said antigen is further modified by reaction of the terminal carboxy group thereof with a nucleophile.

10. An antigen as claimed in Claim 9 in which the said nucleophile is tyrosine ethyl ester or glycine

10 ethyl ester. 10

11. An antigen whenever produced by a process as claimed in any one of Claims 1 to 6.

12. A contraceptive vaccine composition comprising an antigen as claimed in any one of Claims 7 to 11 in a dose amount effective to inhibit the activity of human chorionic gonadotropin.

13. A contraceptive vaccine as claimed in Claim 12 substantially as specifically described herein

15 with reference to the Examples. 15

Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1981. Published by. the Patent Office, 25 Southampton Buildings, London, WC2A 1AY. from which copies may be obtained.